Method of protecting biologically active substances against denaturation

ABSTRACT

A method of protecting a biologically active substance against denaturation, wherein a liquid ( 14 ) containing the active substance and a matrix-forming substance are deposited on a target surface ( 18 ) and dried so as to form a solid amorphous matrix ( 26 ) with the molecules of the active substance embedded therein, wherein an ink jet printer ( 10 ) is used for depositing the liquid ( 14 ) on the target surface ( 16 ) in the form of droplets ( 12 ) having a volume small enough to cause the liquid to dry when it impinges on the target surface ( 16 ) and to be held on the target surface through adhesion.

BACKGROUND OF THE INVENTION

The invention relates to a method of protecting a biologically activesubstance against denaturation, wherein a liquid containing the activesubstance and a matrix-forming substance are deposited on a targetsurface and dried so as to form a solid amorphous matrix with the activesubstance embedded therein.

Numerous active pharmaceutical ingredient molecules and proteinmolecules for therapeutic or prophylactic treatment or diagnosis areknown to be susceptible to denatuation, e.g. to breakdown orirreversible deformation.

In order to be able to keep the active substances stable during storageat ambient temperature, it has been known to suspend the activemolecules in a stabilizing substance which is then transformed into anamorphous solid (glassy) state either by freeze drying or spray drying.

However, freeze drying has the drawback that it requires expensiveequipment and long processing times. Further, the active molecules maybe damaged by the formation of ice crystals in the freezing process.Frequently, a cumbersome post processing of the freeze-dried product isnecessary in order to bring it into a state ready for administration.

On the other hand, spray drying has the disadvantage that a considerableportion of the product remains in the spray dryer, so that the yield ofthe dried active substance is low. Further, the active molecules may bedamaged as a result of exposure to high temperatures during the dryingprocess. Frequently, an additional drying step is necessary in order toreduce the residual moisture.

U.S. Pat. No. 7,354,597 discloses a method wherein microquantities(between 1 nl and 10 pl) of a liquid with the stabilizing substance andthe active substance suspended therein are deposited in microscalereservoirs, i.e. concave-shaped structures on a target surface, and arethen dried with or without a freezing step. Injection and ink jetprinting are mentioned as examples of suitable deposition methods.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method of the typeindicated above, which can be implemented at low costs and with highefficiency and results in products that facilitate the administration ofthe active substance.

According to the invention, this object is achieved by using an ink jetprinter for depositing the liquid on the target surface in the form ofdroplets having a volume small enough to cause the liquid to dry when itimpinges on the target surface and to be held on the target surfacethrough adhesion.

Thus, the substance containing the active substance is simply “printed”onto a substrate which does not have to form microcavities but may havea flat or even convex surface because the liquid droplets, thanks totheir small volume, will dry immediately when they impinge on thesurface and will then stick to that surface through natural adhesion.

The low volume of the droplets has the further effect that most of thevolatile components will evaporate immediately when the droplet hits thetarget surface, so that the substance printed onto the substrate willhave a very low residual moisture and the post-drying step is simplifiedand may be performed in an inline process right after printing, forexample, or no post-drying is needed at all.

More specific optional features of the invention are indicated in thedependent claims.

The rapid drying of the small liquid volume results in the formation ofa glassy solid without requiring a large temperature change rate.Consequently, the entire process may be performed at moderatetemperatures, e.g. at ambient temperature, and will neverthelessreliably result in an amorphous solid which is suitable for stabilizingthe active molecules. More specifically, the volume of droplets shouldbe so small that, when the substrate with the liquid jetted thereon isleft at room temperature for four minutes or less and one then wipeswith a finger over the deposited material, it does not feel sticky andcannot be wiped off.

The substrate that forms the target surface may be made of any suitablematerial and may have any suitable shape and may conveniently beconfigured already for the later administration of the active substance.For example, the substrate may be a patch that is applied to the skin ofa patient so as to release the active molecules through the skin. If thepatch, e.g., a polycarbonate patch, is equipped with transcutaneousneedles to penetrate the skin, the active substance may also be printeddirectly onto the surface of the needles. In other embodiments, thesubstrate may be a transcutaneous needle, a drug dispensing implant or aporous membrane through which a liquid is pressed in order to administerthe active substance. The membrane may be used for treating an infusionliquid or may also form part of a drug dispensing implant. Of course, itis also possible that the active substance is just printed on anysurface from which it will later be washed off in order to dissolve theactive substance in a liquid.

The equipment that is needed for practicing the invention consistsessentially of a low-cost commercial ink jet printer. A piezoelectricink jet printer is preferred, but a thermal (bubble jet) ink jet printermay also be used.

The “biologically active substance” which may be stabilized with themethod according to this invention may be any substance that afteradministration to an animal or human interferes with its normalmetabolism (other then regular digestion), in particular substances thatare either biopolymers such as proteins or polysaccharides or live orkilled micro organisms such as bacteria, viruses and rickettsia, orderivates of these live or killed micro organisms. In particular, thebiologically active substances are antigens for use in a vaccine, i.e. aconstitution for preventing, mitigating or curing an infection with apathogenic micro organism. Other examples are proteins and proteinsub-units, cytokines, other immune-regulating molecules, saponines andthe like. Adjuvants such as alu-gels may also be added.

The liquid to be deposited with the ink jet printer is preferably anaqueous liquid, although other solvents may be used, especially fornon-proteins. The matrix-forming substance may be any commonly usedlyophilisation formulation and should preferably contain a (small)molecule that can provide H-bonds with the proteins of the activesubstance. For example, mono-, di- or oligosaccharides or amino acidsmay be used. Preferably, a polymer such as dextran, gelatine, BSA andthe like may be added in order to increase the glass transitiontemperature Tg. Preferably, the glass transition temperature of thesolid formed on the target surface is in the range from 20 to 60° C. Incertain applications, it may however be as high as 150 or 170° C.

A surfactant may be added to improve the performance of the liquid inthe ink jet printer and/or the deposition or coating characteristics.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments will now be described in conjunction with thedrawings, wherein:

FIG. 1 is a schematic cross-sectional view of a system for practicingthe invention;

FIG. 2 is a schematic view of a production line employing the methodaccording to the invention;

FIG. 3 is a perspective view of an ink jet printer and a substrate usedin the present invention;

FIG. 4 is a cross-sectional view of a product obtained by the methodaccording to the invention;

FIGS. 5(A) and (B) are a side view and a top plan view, respectively, ofa modified printer arrangement for printing onto the substrate shown inFIG. 3; and

FIG. 6 is a side view of yet another printer and substrate arrangement.

DETAILED DESCRIPTION

As is shown in FIG. 1, an ink jet printer 10 is used for jettingdroplets 12 of a liquid 14 onto a target surface 16 of a substrate 18.The liquid 14 may be a water-based liquid containing a sugar such assucrose as matrix-forming substrate, a polymer, and a surfactant. Anactive substance which is to be protected against denaturation issuspended or dissolved in the liquid.

In the inkjet printer 10, the liquid is supplied to a chamber 20 thatopens out into a nozzle 22. A piezoelectric actuator 24 is rigidlysupported in the printer and is separated from the chamber 20 by aflexible sheet 21. When a voltage is applied to the actuator 24, thelatter is deformed abruptly, so that the sheet 21, is deflected into thechamber 20, thereby generating an acoustic pressure wave in the liquid14. The pressure wave propagates towards the nozzle 22 and causes thedroplet 12 to be ejected from the nozzle. The volume of the droplet 12is smaller than 500 pi, preferably smaller than 100 pi or 10 pi and evenmore preferably in the range of 2 to 5 pi.

When the droplet 12 hits the target surface 16, it spreads on thissurface, and since the volume of the droplet is small, thesurface/volume ratio becomes so large that the energy of the droplet issufficient for evaporating almost all of the water contained therein.The non-volatile components (formed mainly by the sugar) form a solidamorphous matrix 26 which sticks to the substrate 18 by natural adhesionand in which the molecules of the active substance are embedded.OH-groups of the sugar replace the OH in the water and form H-bonds withthe molecules of the active substance, so that these molecules arestabilized and protected against degradation.

It will be understood that the print process that has been describedabove can be performed at ambient temperature, e.g. at a temperaturebetween 2 and 30° C., although higher temperatures may be used,depending on the nature of the active substance. In general, a heatingor cooling of the liquid 14 is not necessary, so that the energyconsumption is reduced significantly in comparison to freeze drying orspray drying.

It will further be understood that the ink jet printer 10 may comprise aplurality of nozzle and actuator units arranged in one or more rows, andthese units may fire at a high frequency, so that the matrix 26 on thesubstrate 18 forms a continuous layer when the printhead of the printer10 is moved across the target surface. As in conventional ink jetprinting, the printer may also be controlled to form any desired patternon the matrix.

FIG. 2 shows an example of a production line wherein the substrates 18,e. g. medical patches, are successively placed onto a conveyer belt 28on which the substrates are moved past the ink jet printer 10 in thedirection of an arrow A. The ink jet printer prints a continuous layeror any suitable pattern of amorphous material containing the activesubstance onto the target surface 16 of the substrates. Then, theconveyer belt 28 feeds the substrates through an oven 30 wherein thesubstrates are heated to a moderate temperature to remove residualmoisture from the amorphous material without degrading the activesubstance. Then, the substrates 18 may be taken off the conveyer beltand may immediately be packaged and shipped or stored.

FIG. 3 illustrates a substrate 18′ which takes the form of apolycarbonate patch which has a plurality of subcutaneous (micro-)needles 32 which penetrate the skin of a patient when the patch isapplied. In this example, the ink jet printer 10 is arranged obliquelyrelative to the substrate 18′ so that, when the substrate passes belowthe printer, the droplets may be ejected onto both, the surface of thepatch and the surfaces of the needles 32.

In a preferred embodiment, shown in FIGS. 5(A) and (B), the nozzles 22of two printers 10 are aligned with the rows of needles 32 and theprinters are jointly moved across the substrate 18′ in the direction ofarrow A and are controlled such that the droplets are targeted onto theneedles only. Optionally, two or more printers may be used for obliquelyjetting the droplets onto the needles from different sides, and/or thesubstrate may be scanned in several passes with varying heights of theprinter(s) relative to the needles, so that almost the entire peripheralsurface of the needles may be coated and/or the dose of the activesubstance may be controlled by controlling the length of the needleportion that is being coated.

As an alternative, shown in FIG. 6, a single printer 10 may be arrangedwith its jetting direction normal to the plane of the substrate 18′,i.e. in parallel with the needles, and controlled such that the dropletswill hit the tips of the needles 32 so as to form a coating 34 thereon.

As a result, the active substance can be administered to the patientwith high efficiency. In another embodiment, shown in FIG. 4, thesubstrate may take the form of a porous membrane 18″ with the matrix 26printed on the top target surface 16 thereof. When a liquid, e.g. aninfusion liquid, is pressed through the pores of the membrane frombelow, the bulk material will be dissolved and the active substance willbe released into the liquid. When the membrane 18″ has a regular patternof pores, the printer may be controlled to print only on the edges ofthe pores.

More specific embodiments will be described in the examples below.

EXAMPLE 1

A liquid containing 40% w/t sucrose, and 1% polysorbate 80 in water wasprinted on a substrate made of transparent nitrocellulose with apiezo-type ink jet printer. A solid coating was formed which firmlyadhered to the substrate and was difficult to wipe off. The glasstransition temperature Tg of the coating was determined in a DSC testand was found to be 35° C.

EXAMPLE 2

A liquid containing 20% w/t sucrose, 5% dextran 70 and 2% polysorbate 80was printed under the same conditions as in example 1. The resultingcoating on the substrate was difficult to wipe off and had a Tg of 47°C.

The invention claimed is:
 1. A method of protecting an active substance against denaturation, comprising the steps of: depositing a liquid containing the active substance and a matrix-forming substance on a convex target surface of which at least a part of the target surface is formed by a surface of subcutaneous needles, by at least one ink jet printer in the form of droplets having a volume small enough to cause the liquid to dry when it impinges on the target surface and to be held on the target surface through adhesion, said step of depositing including the steps of: moving at least one said ink jet printer across a regular array of the subcutaneous needles, controlling said at least one said ink jet printer to print onto the needles only, and directing nozzles of said at least one said ink jet printer onto tips of the needles with a jetting direction parallel with a direction of extension of the needles; and drying said deposited liquid so as to form a solid amorphous matrix with molecules of the active substance embedded therein.
 2. The method according to claim 1, wherein the volume of the droplets is less than 500 pl.
 3. The method according to claim 1, wherein the target surface is formed on a medical patch which has the subcutaneous needles.
 4. The method according to claim 1, wherein the at least one ink jet printer is a piezoelectric ink jet printer.
 5. The method according to claim 1, wherein the matrix-forming substance includes a saccharide.
 6. The method according to claim 1, wherein the liquid includes a polymer in an amount to adjust a glass transition temperature Tg of the amorphous matrix to a value between 20 and 200° C.
 7. The method according to claim 1, wherein the liquid includes a surfactant.
 8. The method according to claim 1, wherein the liquid, when it is jetted onto the target surface, has a temperature between 4 and 60° C.
 9. The method according to claim 2, wherein the volume of the droplets is less than 100 pl.
 10. The method according to claim 8, wherein the liquid, when it is jetted onto the target surface, has a temperature between 4 and 50° C.
 11. The method according to claim 9, wherein the liquid, when it is jetted onto the target surface, has a temperature between 4 and 45° C. 